KPC-2基因和外膜蛋白介导的肺炎克雷伯菌对碳青霉烯类药物耐药机制分析  

Analysis of the mechanisms of Klebsiella pneumonia resistance to carbapenem mediated by KPC-2 and outer membrane proteins

作　　者：赵晓杰[1] 康海全[2] 姜飞[1] 施德仕[2] 马萍[1,2] 

机构地区：[1]徐州医学院医学技术学院,江苏徐州221004 [2]徐州医学院附属医院检验科,江苏徐州221002

出　　处：《中国病原生物学杂志》2015年第3期211-214,219,共5页Journal of Pathogen Biology

基　　金：徐州市科技局社会发展项目(No.XZZD1230);徐州医学院附属医院课题(No.2013104033)

摘　　要：目的研究临床分离耐碳青霉烯类肺炎克雷伯菌的耐药机制。方法收集徐州医学院附属医院2013年2-12月临床分离的非重复碳青霉烯类抗菌药物耐药肺炎克雷伯菌株47株并进行鉴定。药敏试验采用琼脂稀释法,采用改良Hodge方法检测碳青霉烯酶,采用EDTA-Na2双纸片协同法检测金属β-内酰胺酶,采用PCR方法检测KPC-2基因,通过肠杆菌基因间重复性一致性序列聚合酶链反应(ERIC-PCR)进行KPC基因分型。采用质粒接合试验确定耐药基因的转移性,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析耐药菌株外膜孔道蛋白的改变情况。结果 47株肺炎克雷伯菌对除多粘菌素和替加环素外的其他抗生素均显示较高的耐药性。改良Hodge试验阳性43株,金属酶检测试验结果均为阴性。ERIC-PCR将44株KPC-2基因阳性肺炎克雷伯菌分为4型。3株分离菌株接合试验获得成功,3株肺炎克雷伯菌外膜孔道蛋白Ompk36的缺失。结论本院分离的碳青霉烯类耐药肺炎克雷伯菌对临床常用抗生素显示较高水平的耐药性,其耐药机制主要是产碳青霉稀酶KPC-2。部分分离菌株存在耐药质粒的水平传播。另外,外膜孔道蛋白的缺失也是导致细菌对碳青霉烯类抗菌药物敏感度降低的重要原因。Objective To investigate the mechanisms of drug resistance in imipenem-resistant Klebsiella pneumoniae isolates from this hospital. Methods Forty-seven strains of carbapenem-resistant K.pneumoniae were collected from Xuzhou Medical College Hospital from February to December 2013 and these strains were identified.Antibiotic susceptibility testing was performed using the disc diffusion method.Carbapenemase production was verified with a modified Hodge test and MBL production was verified with an IPM-EDTA combined disc test.PCR was used to detect resistance genes in the strains.Enterobacterial repetitive intergenic consensus-polymerase chain reaction（ERIC-PCR）was used to analyze molecular epidemiology.Plasmid conjugation experiments were conducted to determine the transferability of resistance genes.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）was used to analyze changes in outer membrane porin. Results The 47 strains had a high level of resistance to routine antibiotics except for colistin and tigecycline.Forty-three strains tested positive for carbapenemase production according to the modified Hodge test,and all47 strains tested negative for MBL production.PCR results indicated that 44 strains carried the KPC-2carbapenemase gene.ERIC-PCR results indicated that the 47 strains of K.pneumoniae fell into 4subtypes,the largest of which was type I.A plasmid conjugation experiment successfully produced carbapenem resistance in 3.Plasmids carrying the KPC-2gene were 41 kb and 48 kb in size.Three strains lacked the outer membrane protein Ompk36. Conclusion Carbapenemresistant K.pneumoniaefrom this hospital had a high level of resistance to routine antibiotics.Forty-seven strains of carbapenem-resistant K.pneumoniae were isolated.The KPC-2gene was the main reason for the bacterium＇s resistance to carbapenems.Horizontal transmission of drug resistance plasmids was evident in some of the isolates.In addition,a lack of porin was also an important cause of carbapenem resistance.

关 键 词：肺炎克雷伯菌 碳青霉烯酶 质粒 接合实验 外膜蛋白 

分 类 号：R378.99[医药卫生—病原生物学]